The protease inhibitor, Bowman-Birk protease inhibitor (BBI), is a low molecular weight (M.sub.r 8,000) double-headed inhibitor of trypsin and chymotrypsin isolated from soybeans. It was first discovered approximately forty years ago and has attracted renewed interest from the scientific research community since the discovery of its potent anticarcinogenic effects in several experimental systems. BBI has been shown to prevent or suppress radiation--and chemical carcinogen-induced malignant transformation in vitro and carcinogenesis in mice, rats and hamsters involving several different organs, tissues and cell types. A soybean preparation enriched in BBI, termed BBI concentrate (BBIC), has recently received investigational new drug status from the U.S. Food and Drug Administration and is currently under early phase clinical evaluation as a potential cancer chemopreventive agent.
The distribution and/or metabolic fate of orally administered BBI has been studied in chicks (Madur et al., Comp. Biochem. Physiol. 1979, 62A, 1057), rats (Yavelow et al., Cancer Res. 1983, 43, 2454) and mice (Persiani et al., Carcinogenesis 1991, 12, 1149; Billings et al., Cancer Lett. 1992, 62, 191) using radio-iodinated BBI and enzyme inhibition assay as the means to quantitate BBI in tissues and body fluids. In mice, orally gavaged BBI was found to be widely distributed in various organs and tissues within 3 hours of BBI administration, and the bulk of BBI was present in the luminal contents of the small and large bowel, urine and feces (Billings et al., Cancer Lett. 1992, 62, 191). While the studies with radioactive BBI have provided important information about the BBI distribution and/or metabolism in these animal models, the same approach is not appropriate for clinical trials due to the concerns about safety and cost. The standard enzyme inhibition assays are not very useful either for measurement of BBI and BBI metabolites in tissue and body fluids since they often do not accurately quantitate specific protease inhibitors, especially in the presence of co-existing protease inhibitors of other types.
High-affinity MAbs to BBI in its native form have been produced by Brandon et al., J. Agric. Food Chem. 1989, 37, 1192. One of the two MAbs, named C238, was found to react with a native structure of BBI sensitive to treatment with thiol-reducing agents that disrupt disulfide bridges. Another MAb, designated C217, recognizes a very heat-labile epitope on native BBI molecules (Brandon, D. L., Protease Inhibitors as Cancer Chemopreventive Agents. (1993) Plenum Press, New York, p. 107). C238 antibody, either alone or in combination with C217, can be used in an ELISA to measure nanograms of purified BBI or BBI in processed soybean food products (Brandon et al., Adv. Exp. Med. Biol. 1991, 289, 321; Brandon, D. L., Protease Inhibitors as Cancer Chemopreventive Agents, Plenum Press, New York, 1993, p. 107); however, neither of these MAbs are capable of detecting BBI metabolites in urine samples collected from humans following oral administration of BBIC. It has been shown, however, in a previous animal study that substantial amounts of orally administered BBI enter the bladder and can be detected in urine (Billings et al., Cancer Lett. 1992, 62, 191).
BBI molecules are rich in disulfide content, with each BBI molecule containing 14 cysteine residues that form 7 intramolecular disulfide bridges which maintain the native structure. It is believed that the disulfide bridges on the native BBI molecule are reduced and possibly re-oxidized or alkylated during metabolism in the presence of thiol modifying agents, such as glutathione and other small molecular weight thiols, in tissues and body fluids. The inability of C238 to detect BBI metabolites in human urine samples after BBI administration indicates that at least some of the disulfide bonds on BBI molecules are broken in vivo. It has now been found that immunizing animals with reductively modified BBI results in production of MAbs reactive with BBI metabolites in tissues and body fluids. These MAbs are useful in monitoring exposure levels to BBI during clinical studies.